Nitric oxide (NO) is believed to function as a CNS neurotransmitter and neuromodulator, where it is involved in learning, development, and neurotoxicity under some circumstances. However, its mechanisms of action are poorly understood, in part, because it is a free radical that readily passes across membranes, but also because there are presently few well-defined central neuron preparations for its study where the pre- and postsynaptic neurons are experimentally accessible. We propose to study well defined synaptic pairs, consisting of identified cerebral neuron C2, which contains the enzyme nitric oxide synthase and uses NO as an orthograde co-transmitter to its follower neurons, C4 and MCC, in the mollusc Aplysia. These neurons are part of the feeding behavior neural circuit that is richly modulated, and in which many motor acts must be specifically patterned, smoothly coordinated and appropriately timed. The objectives of this proposed research are to determine the neurotransmitter and neuromodulatory mechanisms. The synaptic potentials between the identified neurons will be recorded using membrane patch methods and manipulated using inhibitors and activators of the two principal enzymes that are believed to be involved, nitric oxide synthase in the presynaptic neuron and guanylyl cyclase in the postsynaptic neurons. The neurons will be isolated in cell culture in order to determine the release, diffusion and reception of NO produced by the isolated neurons or by release from caged NO compounds injected in the neurons. Follower neurons will serve as sensors of released NO, by monitoring changes in membrane current and conductance. In culture, calcium imaging will be used to determine the calcium dependence of NO release. Changes in cGMP concentration, during synaptic transmission or in response to co-transmitters NO and histamine, of guanylyl cyclase will be measured using immuno-cytochemistry and radioimmune assay. The evidence gathered from these experiments will provide detailed information about the basic mechanisms of orthograde synaptic transmission mediated by NO and neuromodulation of excitability and synaptic input from other neurons in Aplysia. History has shown that basic mechanisms discovered in molluscs, e.g. action potentials and synaptic plasticity, hold true in mammalian nervous systems as well.